CN105699657B - A method for detecting Vimentin in peripheral blood of renal cancer patients - Google Patents

Abstract

The invention discloses a method for detecting Vimentin in the peripheral blood of patients with renal cancer: using a membrane filtration device to separate and obtain CTCs in the peripheral blood of advanced or recurrent renal cancer patients whose tissue samples cannot be obtained, and using cell wax block technology to make thin-layer slices, and then detecting Vimentin expresses the situation. Through this technical method, the expression of Vimentin in patients with renal cancer can be detected without taking renal cancer tissue. The technology is minimally invasive or even non-invasive, and can detect in real time.

Description

A method for detecting Vimentin in peripheral blood of renal cancer patients

technical field

The invention relates to a new method for detecting Vimentin, in particular to a method for detecting Vimentin in renal cancer patients through peripheral blood.

Background technique

Vimentin (Vimentin) is an important intermediate filament protein, and its main biological functions are: maintaining the shape of cells and organelles, promoting cell adhesion and migration, participating in mitosis and cell differentiation, wound healing, signal transduction, transplantation immunity and cell Apoptosis etc. It is mainly expressed in mesenchymal tissue and can be used as a specific biomarker to identify the origin of tumor cell differentiation in histopathological diagnosis. Studies have shown that the high expression of Vimentin is closely related to the low degree of differentiation, strong invasiveness and easy metastasis of renal cancer cells. Vimentin can be used as an indicator for the stage, grade and prognosis of renal cancer. It is a targeted drug or cytokine after renal cancer surgery. Adjuvant therapy provides the basis and therapeutic targets, therefore, it is very important to correctly detect the expression of Vimentin in patients with RCC.

There are two commonly used Vimentin detection methods at present. One is an immunohistochemical method, which determines the status of Vimentin by detecting the expression of Vimentin protein in renal cancer tissue samples. Another method is Western blot, which has higher specificity and is currently being used more and more in clinical practice. These two detection methods are based on renal cancer tissue as the detection object, especially for patients undergoing surgical resection of renal cancer. However, for some RCC patients who lack surgical indications or have surgical contraindications (such as patients with advanced RCC), the evaluation of Vimentin status becomes difficult due to the unresectable tumor tissue and the possibility of tumor dissemination due to fine-needle aspiration.

Contents of the invention

Circulating tumor cells (Circulating tumor cells, CTCs) are tumor cells that come off from solid tumors and enter the peripheral blood circulation. They exist in various malignant tumors such as renal cancer, breast cancer, prostate cancer, and colorectal cancer. The prognosis of tumor patients, especially those with advanced tumors, and the selection of appropriate individualized treatment have important clinical significance. Because CTC detection is minimally invasive and real-time, it is called "liquid biopsy".

In order to overcome the inadequacy of taking tissue samples from patients with advanced renal cancer and the inability to evaluate the Vimentin status of patients, the present invention provides a method for detecting peripheral blood Vimentin in patients with renal cancer: using a membrane filtration device to separate and obtain advanced or recurrent patients who cannot obtain tissue samples CTCs in the peripheral blood of patients with kidney cancer were thin-layer sliced using cell wax block technology, and then the expression of Vimentin was detected.

The technical scheme that the present invention adopts is as follows:

1. Using a membrane filtration device to separate and obtain CTCs in the peripheral blood of patients with advanced or recurrent renal cancer who cannot obtain tissue samples:

1. Collect peripheral blood from patients with advanced renal cancer: 5ml from median cubital vein;

2. Peripheral blood sample pretreatment: Dilute the collected peripheral blood sample 10 times, the dilution liquid composition: 1mmol/l EDTA+0.1%BSA, after dilution, add paraformaldehyde to fix the peripheral blood sample for 10 minutes, and fix the final concentration to 0.25%;

3. Use the membrane filtration tumor cell separation device to filter peripheral blood samples to separate and obtain peripheral blood circulating tumor cells: add the pretreated peripheral blood sample to the blood sample container of the membrane filtration tumor cell separation device, and let it naturally filter by gravity;

4. After the filtration, remove the filter from the membrane filtration device for separating tumor cells, add circulating tumor cell staining solution to the filter, stain for 2 minutes, rinse with PBS buffer, remove the filter membrane with fine tweezers, and place it on a glass slide. On slices, dry and observe under a microscope to determine the presence or absence of CTCs.

2. Using cell wax block technology to make thin-layer slices:

1. Decolorization: Remove the filter membrane with CTC on the above glass slide, soak in the decolorization solution for 4-6 hours, and remove the CTC staining solution. The decolorization solution is: 95% alcohol and 100% xylene Mix with a volume ratio of 1:1.

2. Wrapping: After soaking, take out the filter membrane and wrap it with absorbent paper;

3. Fixing: Put the wrapping in 10% neutral formalin and fix it for 4-6 hours;

4. Embedding in wax: After the fixation, take out the wrapper, put it in a paraffin embedding box after dehydration, and embed in wax to make a paraffin block of cells;

5. Thin-layer section: Use a microtome to slice the cell paraffin block continuously to make a thin-layer section with a thickness of 4 μm.

3. Detection of Vimentin expression:

1. Thin-layer slices were dewaxed and hydrated in the staining vat according to the following steps: soak in xylene solution for 3 times, each time for 10 minutes; soak in absolute ethanol solution for 3 times, each time for 5 minutes; soak in 95% ethanol solution for 5 minutes minutes; soak in 85% ethanol solution for 5 minutes; soak in 75% ethanol solution for 5 minutes; soak in distilled water twice, 3 minutes each; soak in PBS (PH=7.4) 3 times, 3 minutes each;

2. Restore tissue antigens by using citrate buffer high temperature and high pressure antigen repair method: take 800-1500ml of citrate buffer solution with pH=6.0 in a pressure cooker, heat it on high heat until boiling, and place the dewaxed and hydrated tissue slices in Put it into the boiling buffer solution on the high-temperature stainless steel slice rack, continue to heat the pot cover until the steam is sprayed, and start timing. After 1-2 minutes, leave the pressure cooker from the heat source, cool to room temperature, take out the slides, and rinse them twice with distilled water , followed by washing twice with PBS of pH=7.2-7.4, each time for 3 minutes;

3. Incubate with 3% H ₂ O ₂ deionized water for 5 minutes to block endogenous peroxidase, wash with PBS 3 times, 2 minutes each time;

4. Add Vimentin primary antibody dropwise, incubate at room temperature or 37°C for 1-2 hours or overnight at 4°C, wash with PBS 3 times, each time for 2 minutes;

5. Add universal IgG antibody dropwise, incubate at room temperature or 37°C for 15 minutes, wash with PBS 3 times, 2 minutes each time;

6. Apply DAB solution for color development: shake off the PBS solution, add 2 drops or 100 μl of freshly prepared DAB solution to each slice, and observe under a microscope for 3-10 minutes;

7. Rinse with distilled water, counterstaining, dehydration, transparent mounting: rinse with distilled water twice, 3 minutes each time; counterstain with hematoxylin for 1 minute; 0.1% hydrochloric acid alcohol differentiation; 0.1% ammonia water to turn blue; 50%, 70%, 85% , 95%, dehydrated and dried with absolute ethanol; transparent xylene, sealed with neutral resin;

8. Cell pathology experts read the slides and judge the expression of Vimentin according to the degree of cell staining.

The device for separating tumor cells by membrane filtration described in the step includes a filter, a blood sample container, a waste liquid tank, and an iron stand. The iron stand is provided with a base, a stand and a bracket, and the blood sample container is arranged on the upper part of the iron stand through the bracket. Below the blood sample container is a filter, which is connected to the waste liquid tank through the infusion set, and the waste liquid tank is arranged on the base.

The filter includes an upper port of the filter, a filter membrane, a platform for carrying the filter membrane and a lower port of the filter, the filter membrane is placed on the platform for carrying the filter membrane; the upper port of the filter is connected to the blood sample container, and the lower port of the filter is connected to the waste liquid tank through the infusion set.

The filter membrane is made of hydrophobic material, and is evenly covered with filter holes with a diameter of 10 microns.

The beneficial effects of the present invention are: through the technical method, the expression of vimentin in patients with renal cancer can be detected without sampling renal cancer tissue. The technology is minimally invasive or even non-invasive, and can detect in real time.

Description of drawings

Fig. 1 is the structural representation of membrane filtration device of the present invention;

Fig. 2 is the schematic sectional view of the structure of the filter of the membrane filtration device of the present invention;

Fig. 3 is the structural representation of the filter membrane of membrane filtration device of the present invention;

Figure 4 is an image of circulating tumor cells obtained from the peripheral blood of a patient with advanced renal cancer;

Figure 5 is an image of Vimentin immunohistochemical staining of circulating tumor cells in peripheral blood of patients with advanced renal cancer.

In the figure: 1 iron stand, 2 blood sample container, 3 filter, 4 infusion set, 5 waste liquid cylinder, 6 filter upper port, 7 filter membrane, 8 filter membrane platform, 9 filter lower port, 10 filter hole, 11 base, 12 stands, 13 brackets.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

An example of using this technique to separate, acquire and identify peripheral blood circulating tumor cells from 8 patients with advanced renal cancer (simultaneously detect 8 normal samples as negative controls).

1. Using a membrane filtration device to separate and obtain CTCs in the peripheral blood of patients with advanced or recurrent renal cancer who cannot obtain tissue samples:

Collect 5ml of peripheral blood from each sample, dilute the peripheral blood with 45ml diluent (ingredient: 1mmol/l EDTA+0.1%BSA), and then add 3ml of 4% paraformaldehyde to fix the diluted blood sample for 10 minutes.

At a fixed interval, assemble the membrane filter device: as shown in Figure 1, Figure 2, and Figure 3, the filter device consists of a filter 3, a filter membrane 7, a blood sample container 2, a waste liquid tank 5, and an iron stand 1.

Wet the filter 3 with 10ml of PBS, then add the fixed peripheral blood sample into the blood sample container 2 of the membrane filtration device, let it filter naturally by gravity, and CTCs are trapped on the filter membrane 7 .

The diameter of tumor cells is generally greater than 15 microns, while the diameter of blood cells (including red blood cells and white blood cells) is generally less than 10 microns. Therefore, when the peripheral blood containing CTCs is filtered, blood cells can be filtered because the diameter is smaller than the filter hole, while CTCs can be filtered because the diameter is larger than the filter hole. The pores are trapped on the filter membrane.

After the filtration, remove the filter 3 from the filter device, open and remove the upper port 6 of the filter, add circulating tumor cell Diff staining solution to the filter 3, and stain for 2 minutes.

Rinse the filter 3 with PBS buffer solution, remove the filter membrane 7 with fine tweezers, and place it on a glass slide with the cell facing up.

After the filter membrane 7 is properly dried, it is observed under a microscope to determine whether there are CTCs.

Test results: No circulating tumor cells were found in 8 healthy volunteers; circulating tumor cells were detected in 4 patients with advanced renal cancer (Table 1). The positive rate of this test is 50%.

2. Using cell wax block technology to make thin-layer slices:

1. Decolorization: remove the filter membrane 7 with CTC on the slide glass, soak in the decolorization solution for 4-6 hours, and remove the CTC staining solution. The decolorization solution is: 95% alcohol and 100% xylene by volume Mixed at a ratio of 1:1.

2. Wrapping: After soaking, take out the filter membrane and wrap it with absorbent paper;

3. Fixing: Put the wrapping in 10% neutral formalin and fix it for 4-6 hours;

4. Embedding in wax: After the fixation, take out the wrapper, put it in a paraffin embedding box after dehydration, and embed in wax to make a paraffin block of cells;

5. Thin-layer section: Use a microtome to slice the cell paraffin block continuously to make a thin-layer section with a thickness of 4 μm.

3. Detection of Vimentin expression:

1. Dewax and hydrate the CTC wax block thin-layer slices in the dyeing vat according to the following steps: soak in xylene solution for 3 times, each time for 10 minutes; soak in absolute ethanol solution for 3 times, each time for 5 minutes; 95% Soak in ethanol solution for 5 minutes; soak in 85% ethanol solution for 5 minutes; soak in 75% ethanol solution for 5 minutes; soak in distilled water twice, 3 minutes each; soak in PBS (PH=7.4) 3 times, 3 minutes each;

2. Restore tissue antigens by using citrate buffer high temperature and high pressure antigen repair method: take 800-1500ml of citrate buffer solution with pH=6.0 in a pressure cooker, heat it on high heat until boiling, and place the dewaxed and hydrated tissue slices in Put it into the boiling buffer solution on the high-temperature stainless steel slice rack, continue to heat the pot cover until the steam is sprayed, and start timing. After 1-2 minutes, leave the pressure cooker from the heat source, cool to room temperature, take out the slides, and rinse them twice with distilled water , followed by washing twice with PBS of pH=7.2-7.4, each time for 3 minutes;

3. Incubate with 3% H ₂ O ₂ deionized water for 5 minutes to block endogenous peroxidase, wash with PBS 3 times, 2 minutes each time;

4. Add Vimentin primary antibody dropwise, incubate at room temperature or 37°C for 1-2 hours or overnight at 4°C, wash with PBS 3 times, each time for 2 minutes;

5. Add universal IgG antibody dropwise, incubate at room temperature or 37°C for 15 minutes, wash with PBS 3 times, 2 minutes each time;

6. Apply DAB solution for color development: shake off the PBS solution, add 2 drops or 100 μl of freshly prepared DAB solution to each slice, and observe under a microscope for 3-10 minutes;

7. Rinse with distilled water, counterstaining, dehydration, transparent mounting: rinse with distilled water twice, 3 minutes each time; counterstain with hematoxylin for 1 minute; 0.1% hydrochloric acid alcohol differentiation; 0.1% ammonia water to turn blue; 50%, 70%, 85% , 95%, dehydrated and dried with absolute ethanol; transparent xylene, sealed with neutral resin;

8. Cell pathology experts read the slides and judge the expression of Vimentin according to the degree of cell staining.

Example Test results: no circulating tumor cells were found in 8 healthy volunteers; circulating tumor cells were detected in 4 patients with advanced renal cancer (Table 1). The positive rate of this test was 50%. Immunohistochemistry was used to confirm the expression of Vimentin in the detected circulating tumor cells, and compared with the results of Vimentin in gross specimens of renal cell carcinoma to guide the diagnosis of advanced renal cell carcinoma and the determination of the efficacy of targeted therapy. Cancer targeted therapy provides new ideas.

Figure 4 shows the images of circulating tumor cells obtained from the peripheral blood separation of patients with advanced renal cancer. The nuclei are large, the nuclei are irregular in shape, and the ratio of nuclei to cytoplasm is high.

Figure 5 shows the Vimentin immunohistochemical staining image of peripheral blood circulating tumor cells in patients with advanced renal cancer (judging the positive degree according to its staining distribution and intensity: judging the positive degree according to its staining distribution and intensity, the cell staining accounts for less than 10% of the cells as (- ), coloring 10%-25% is (+), coloring 25%-50% is (++), coloring is greater than 50% (+++)).

Table 1 embodiment detection result

Claims (4)

1. A detection method for non-diagnostic purpose of peripheral blood Vimentin in patients with kidney cancer, characterized in that the detection method steps are as follows: use a membrane filtration device to separate and obtain CTCs in the peripheral blood of patients with advanced kidney cancer who cannot obtain tissue samples, and use cell wax Block technology to make thin-layer slices, and then detect the expression of Vimentin; The steps for separating and obtaining CTCs in the peripheral blood of patients with advanced renal cancer whose tissue samples cannot be obtained by using a membrane filtration device are as follows: 1) Collect peripheral blood from patients with advanced renal cancer: 5ml from median cubital vein; 2) Peripheral blood sample pretreatment: Dilute the collected peripheral blood sample 10 times, the diluent composition: 1mmol/l EDTA+0.1%BSA, after dilution, add paraformaldehyde to fix the peripheral blood sample for 10 minutes, and fix the final concentration to 0.25%; 3) Using the membrane filtration tumor cell separation device to filter peripheral blood samples to separate and obtain peripheral blood circulating tumor cells: add the pretreated peripheral blood sample to the blood sample container (2) of the membrane filtration tumor cell separation device, allowing it to naturally filter by gravity; 4) After the filtration, remove the filter (3) from the device for separating tumor cells by membrane filtration, add circulating tumor cell staining solution to the filter (3), stain for 2 min, rinse with PBS buffer, and remove the filter with fine tweezers. The film (7) is placed on a glass slide, and observed under a microscope after drying to determine whether there are CTCs; The steps of using the cell wax block technique to make thin-layer slices are as follows: 1) Decolorization: remove the filter membrane (7) on the above-mentioned glass slide, soak in the decolorization solution for 4-6 hours, and remove the CTC staining solution. The decolorization solution is: 95% alcohol and 100% xylene Volume ratio 1:1 mixing; 2) Wrapping: Take out the filter membrane (7) after soaking and wrap it with absorbent paper; 3) Fixing: place the wrapping in 10% neutral formalin, and fix it for 4-6 hours; 4) Embedding in wax: take out the wrapper after fixation, dehydrate and place in a paraffin embedding box, and embedding in wax to make a paraffin block of cells; 5) Thin-layer sectioning: use a microtome to slice the cell paraffin blocks continuously to make thin-layer sections with a thickness of 4 μm; The described detection Vimentin expression, the steps are as follows: 1) Carry out dewaxing and hydration of CTC wax block thin-layer sections in the staining vat according to the following steps: soak in xylene solution for 3 times, each time for 10 minutes; soak in absolute ethanol solution for 3 times, each time for 5 minutes; 95% Soak in ethanol solution for 5 minutes; soak in 85% ethanol solution for 5 minutes; soak in 75% ethanol solution for 5 minutes; soak in distilled water twice, each time for 3 minutes; PH=7.4 PBS soak for three times, each time for 3 minutes; 2) Restore the antigen by using citrate buffer high temperature and high pressure antigen retrieval method: take 800-1500ml of citrate buffer solution with pH = 6.0 in a pressure cooker, heat on high heat until boiling, and place the dewaxed and hydrated slices in a high temperature resistant Put it on the stainless steel slicing rack, put it into the boiling buffer solution, cover the pot and continue to heat until the steam spray starts to count. After 1-2 minutes, leave the pressure cooker from the heat source, cool to room temperature, take out the slide, and rinse it twice with distilled water , followed by washing twice with PBS of pH=7.2-7.4, each time for 3 minutes; 3) Incubate in 3% H ₂ O ₂ deionized water for 5 minutes to block endogenous peroxidase, wash with PBS 3 times, 2 minutes each; 4) Add Vimentin primary antibody dropwise, incubate at room temperature or 37°C for 1-2 hours or overnight at 4°C, wash with PBS 3 times, 2 minutes each time; 5) Add universal IgG antibody dropwise, incubate at room temperature or 37°C for 15 minutes, wash with PBS 3 times, 2 minutes each time; 6) Apply DAB solution for color development: shake off the PBS solution, add 2 drops or 100 μl of freshly prepared DAB solution to each section, and observe under a microscope for 3-10 minutes; 7) Rinse with distilled water, counterstain, dehydrate, and transparent mount: rinse with distilled water twice, 3 minutes each time; counterstain with hematoxylin for 1 minute; 0.1% hydrochloric acid alcohol differentiation; 0.1% ammonia water to turn blue; 50%, 70%, 85% , 95%, dehydrated and dried with absolute ethanol; transparent xylene, sealed with neutral resin; 8) Cytopathology experts read the slides and judge the expression of Vimentin according to the degree of cell staining. 2. the non-diagnostic detection method of renal cancer patient's peripheral blood Vimentin according to claim 1, is characterized in that, described membrane filtration device comprises filter (3), blood sample container (2), waste liquid cylinder (5) And iron frame platform (1), described iron frame platform (1) is provided with base (11), stand (12) and support (13), and described blood sample container (2) is arranged on iron frame platform (1) by support (13) ), the filter (3) is below the blood sample container (2), and the filter (3) is connected to the waste liquid tank (5) through the infusion set (4), and the waste liquid tank (5) is arranged on the base (11). 3. the detection method of non-diagnostic purpose of renal cancer patient's peripheral blood Vimentin according to claim 2, is characterized in that: described filter (3) comprises filter upper port (6), filter membrane (7), carries filter membrane platform (8) and the lower port of the filter (9), the filter membrane (7) is placed on the filter membrane platform (8); the upper port of the filter (6) is connected to the blood sample container (2), and the lower port of the filter (9) passes through the infusion set ( 4) Connect the waste liquid cylinder (5). 4. the non-diagnostic detection method of peripheral blood Vimentin of kidney cancer patients according to claim 3, is characterized in that: described filter membrane (7) is made of hydrophobic material, is evenly covered with the filter that diameter is 10 microns on it. hole (10).